This project will investigate biofilm formation by a widespread gram-negative aerobic bacterium, Variovorax paradoxus. The specific aims of this project will be to understand the genetic and physiologic basis for extracellular polymeric substance (EPS) production and how this biosynthetic pathway influences the formation of stable aggregated structures (biofilms) on surfacs. The project will identify the genes and corresponding products essential for EPS biosynthesis by insertional mutagenesis, and will simultaneously identify mutants that are no longer capable of forming biofilms. Further work will characterize the roles of various genes in EPS biosynthesis and biofilm formation by complementation of the knocked out genes, and analysis of gene expression in the developing film using a promoterless GFP construct. Variovorax paradoxus is widespread in the environment, and has previously been identified as a degrader of xenobiotic contaminants. Well developed genetic and physiological systems utilizing this microbe may thus be utilized in bioremediative settings to remove contaminants from polluted sites. The biofilm lifestyle has been strongly implicated in the course of many gram negative bacterial infections, as well as in antibiotic resistance. It is therefore extremely important to use multiple model systems for biofilm formation that allow for the discovery of potential therapeutic targets that might inhibit biofilm formation in a variety of organisms. The EPS biosynthesis pathways are strongly linked to biofilm formation and pathogenesis in a number of systems. By identifying genes essential to biofilm formation and EPS synthesis in V. paradoxus, and comparison to the available sequenced genomes of related plant and human pathogens, we may identify such targets in the cell for chemotherapy.